1. Field of the Invention
This invention relates to a method for detecting a target substance by utilizing plural reagents which can form a reaction system causing a change based on an interaction mediating the target substance, for example, a method useful for detection and identification of a desired base sequence of nucleic acids (DNA or RNA) of virus, microbes, animals, plants, and human beings, and detection of mutation in base sequences, and detection of various substances with immune reactions such as immunoassay.
2. Related Background Art
With advances of analytical techniques of nucleic acids, various kinds of mutation genes have been found, and hereditary diseases caused by gene mutations have been gradually clarified. Gene mutations include partial base deletion and base point mutation, which have been found to cause protein mutation, resulting in various kinds of symptoms. Presently, the hereditary diseases were mainly determined by assays utilizing enzymes or immunological methods utilizing antibodies after symptoms appeared. However, it is said that detection of mutant gene is important at an early stage before severe symptoms appear.
DNA diagnosis is applicable not only for detection of genes of human beings, but for identification of infecting bacteria.
Conventionally, bacterial strain has been identified based on the analogy of morphological and biochemical properties in separated bacteria. This method has the following disadvantages; it requires much time for culture; the same properties can be differently judged by utilizing different methodologies; and different results are obtained for identification when some properties are more predominantly considered than other ones in analysis.
Recent years, DNA-DNA hybridization method and DNA-RNA hybridization method have been tried to detect and identify pathogenic bacteria in bacterial infections. The methods comprise the following steps; extraction of nucleic acids (DNA or RNA) from bacteria, selection of specific part of nucleic acids from bacteria, detection of a base sequence of high homology with the base sequence of the specific part in test nucleic acid samples according to hybridization, and judgement of the subjective bacteria in the samples.
A new method for detecting a specific base sequence of nucleic acid, PCR method, is also utilized. This method comprises the following steps; selection of a specific sequence in a target nucleic acid, preparation of primers required for amplification of the specific sequence, conduction of PCR utilizing the target nucleic acid as a template, detection of the amplified specific sequence, and detection of the target nucleic acid. Use of PCR method improves sensitivity for in detection of nucleic acids. Hybridization is therefore replaced with PCR for detection of nucleic acids in various kinds of fields.
However, PCR method can detect a specific sequence only when a subjective specific base sequence is apparent and an optimal primer is amplified. Primer often combines with a target nucleic acid in non-specific manner, and such sequences as essentially not to be amplified are often formed. Although abnormal gene such as deletion in a target nucleic acid can be determined by analyzing the length of the nucleic acid, abnormal genes such as point mutation which does not change the nucleic acid in length can be determined by no means.
Hybridization is not completely replaced with PCR, but it is also utilized as a method for easily detecting gene.
In hybridization, a probe DNA and a target DNA are combined through a hydrogen bond at their sequence parts which are complementary with each other, in order to form a hybrid. Since a probe cannot be combined with a nucleic acid having a complementary sequence at higher temperatures, and a probe inversely combines with a nucleic acid in non-specific manner in lower temperatures, optimal reaction temperature and ionic strength need to be selected in order to accurately form between the complementary sequences. Further, to make hybridization more accurate, such probes as combining in non-specific manner or mismatching need to be washed by reducing the salt concentration of the hybridization solution or by elevating the temperature of the solution. Therefore, many trials and errors are needed to determine adequate conditions for reaction and washing.
For gene diagnosis, conditions for hybrid forming reaction and washing should be determined more precisely to exclude even a mismatch of one base pair.
Conventionally, hybridization reaction was conducted by immobilizing target nucleic acids on a carrier such as nitrocellulose. The hybridization requires many complicated manipulations, and a novel technique such as hybridization in a solution is expected to develop to make manipulations easy. In the hybridization with no immobilization of nucleic acid immobilization, the largest problem is how to discriminate the objective probes combined with target nucleic acids from excessive probes which are not combined (B/F separation). Moreover, it is important to determine adequate conditions for reaction and washing so that non-specific absorption or mismatch of probes may be excluded for this hybridization as well as for the conventional hybridization utilizing immobilized nucleic acids.
Several methods utilizing fluorescence depolarization are provided in order to detect hybrid of target nucleic acid and probe no B/F separation (Japanese Patent Appln. Laid-Open Nos. 2-295496 and 2-75958). In these methods, a fluorescent-labelled single stranded DNA probe is contacted with DNA in a sample to form a double stranded DNA, fluorescence polarizations before and after the formation of the double stranded DNA are measured, and the resulting variation is evaluated in order to determine whether a base sequence corresponding to the probe base sequence resides in the sample DNA or not. This method is based on the principle that fluorescent substance combined with the single stranded probe get hard to move in the formation of the double stranded DNA, resulting in elevated fluorescence anisotropy.
Although the methods need not B/F separation, they have the following defects: contaminants such as protein in a sample can adsorb to the probe DNA in non-specific manner to increase background for hybrid detection; complicated manipulations are therefore required to exclude the contaminants in advance, non-specifically absorbed probe DNA and pseudo-hybrid due to base mismatch should be excluded in advance when different solution system is utilized, and the concentration of the probe DNA should be almost the same as that of the target DNA to precisely measure the variation of fluorescence.
Gardullo et al. provide three methods for detection of hybrid utilizing energy transfer (Proc. Nalt. Acad. Sci. USA, 85, 8790-8794). All of the methods utilize fluorescein and acridine orange as an energy donor, and rhodamine as an energy acceptor, and conduct the following hybridizations; (i) hybridization of oligonucleotide labelled with fluorescein at its 5' terminal and its complementary oligonucleotide labelled with rhodamine at its 5' terminal, (ii) hybridization of oligonucleotide labelled with fluorescein at its 5' terminal and its complementary DNA, and (iii) hybridization of oligonucleotide labelled with rhodamine and its complementary DNA in the presence of acridine orange. The hybridizations are phenomena that excited energy of the donor is transferred to the neighboring acceptor when excited light and fluorescence in the neighboring fluorescence chromophore are overlapped, leading to shortened lifespan of the donor, quenching of the donor fluorescence, elevated fluorescence strength, etc. The methods are epochal in that hybridization can be detected in a solution and a series of complicated manipulations for immobilization are omitted. However, their sensitivities are lower in several orders compared to the conventional hybridizations. Therefore, modification of the fluorescence chromophore and drastic progress of a detection system have been expected for practical use.
Recently, it is reported that double stranded DNA are mixed with dyes of two kinds in free condition and charge transfer is detected through DNA between the dyes (J. Ame. Chem. Sco., 1992, 114, 3656-3660). In the study, an electron donor having fluorescence (ethidium bromide or acridine orange) is irradiated with lights of wavelengths corresponding to the respective excited wavelengths, and the fluorescence light strength is then reduced in the presence of another dye (an electron acceptor: N,N-dimethyl-2,7-diazapyrenium dichloride). It is considered that both of the dyes are intercalator, and the electron is transferred from the electron donor through DNA double helix to the electron acceptor. However, the degree of the transfer is too low to detect hybrid. Also, the dyes have fluorescence even in free condition, and background of the dye fluorescence should be always considered, even when the variation of the fluorescence strength is detected.
As described above, complicated manipulations are needed to prevent or exclude non-specific adsorption of probes and mismatch of combination, not only for the conventional hybridization utilizing immobilization of target nucleic acids but for the methods such as fluorescence depolarization which require no B/F separation. Moreover, optimal conditions for the manipulations are varied according to the length of a probe and a base sequence for use, and the conditions need to be evaluated and determined for each case. Specifically, the conditions for hybridization need to be determined for each case by considering the possible mismatch based on the fact that the base position of mismatched probe can be an important factor affecting the stability of hybrid, and some mismatched hybrids cannot be excluded dependently on the mismatched position.
The detection systems in a solution utilizing charge transfer or energy transfer are easier for manipulation than the conventional methods which require immobilization of target nucleic acids and B/F separation. However, they are not practically applicable because of low sensitivity. Since the conventionally used dyes have fluorescence even in free condition, it is difficult to decide that the variation of the detected fluorescence strength is caused by an interaction of the energy donor and the energy acceptor (i.e. the electron donor and the electron acceptor) through DNA, by a simple quenching of the solution, or by effects of contaminants.